It is now generally accepted that many important cellular phenotypes, from disease states to metabolite overproduction, are affected by many genes. Yet, most cell and metabolic engineering approaches rely almost exclusively on the deletion or over-expression of single genes due to experimental limitations in vector construction and transformation efficiencies. These limitations preclude the simultaneous exploration of multiple gene modifications and confine gene modification searches to restricted sequential approaches where a single gene is modified at a time.
U.S. Pat. No. 5,686,283 described the use of a sigma factor encoded by rpoS to activate the expression of other bacterial genes that are latent or expressed at low levels in bacterial cells. This patent did not, however, describe mutating the sigma factor in order to change globally the transcription of genes.
U.S. Pat. No. 5,200,341 provides a mutated rpoH gene identified as a suppressor of a temperature sensitive rpoD gene by selection of temperature-resistant mutants of a bacterial strain having the temperature sensitive rpoD gene. No mutagenesis of the bacteria was undertaken, nor was the suppressor strain selected for a phenotype other than temperature resistance. When the mutant rpoH gene is added to other bacteria that are modified to express heterologous proteins, the heterologous proteins are accumulated at increased levels in the bacteria.
U.S. Pat. No. 6,156,532 describes microorganisms that are modified by introduction of a gene coding for a heat shock protein and a gene coding for a sigma factor (rpoH) that specifically functions for the heat shock protein gene to enhance expression amount of the heat shock protein in cells. The modified microorganisms are useful for producing fermentative products such as amino acids. The sigma factor used in the microorganisms was not mutated.
Directed evolution has been applied to microorganisms by shuffling of bacterial genomes for antibiotic (tylosin) production by Streptomyces (Zhang et al., Nature, 415, 644-646 (2002)) and acid tolerance of Lactobacillus (Patnaik et al., Nature Biotech. 20, 707-712 (2002)). These methods did not target mutations in any specific gene or genes, but instead non-recombinantly shuffled the genomes of strains having a desired phenotype using protoplast fusion, followed by selection of strains having improvements in the desired phenotype.